Salmonellacide composition-containing propionic acetic and butyric or phosphoric acids

ABSTRACT

Salmonellacides for use in animal by-product meal materials comprise propionic acid alone and in combination with such complementary acid materials as acetic, butyric, citric, lactic and phosphoric acids.

United States Patent Khan et al. 1451 Oct. 10, 1972 SALMONELLACIDECOMPOSITION- [5Q] V lleferences Cited CONTAINING PROPIONIC ACETIC UNITEDSTATES PATENTS g gg OR PHOSPHORIC 1,589,866 6/ 1926 Siegler et a]...424/317 X 2,276,234 3/1942 Jone ..47/58 1 Inventors: Mahmoud Khan,Chwago; 2,688,585 9/1954 Wilder et al. ..l67/58 Myron Kammay, Oak Forest3,115,409 12/1963 l-lallinan et a1. ..99/7 bOth 0f 3,219,453 11/1965Bogdonofi' et al. ..99/4 [73] Assignee: Fats and Proteins ResearchFoundation, lnc., Des Plaines, 111. Primary ExaminerAlbert T. Meyers 1Assistant Examiner--Allen J. Robinson 22 F1 d: A 1118 1969 1 1 6 prAttorney-Olson, Trexler, Wolters & Bushnell [21] Appl. No.: 817,558

ABSTRACT [52] U.S.Cl. ..424/128, 99/2, 424/317 Salmonellacides for usein animal byproduct meal materials comprise propionic acid alone and incombination with such complementary acid materials as acetic, butyric,citric, lactic and phosphoric acids.

SALMONELLACIDE COMPOSITION- CONTAINING PROPIONIC ACETIC AND BUTYRIC ORPHOSPHORIC ACIDS BACKGROUND OF THE INVENTION This invention relatesgenerally to the preservation of foodstuffs and more particularly toadditive materials that are bactericidally effective against Salmonellaorganisms in dry meal products.

Among the pathogenic bacteria of major concern to food and feedprocessors is the genus Salmonella. Unlike certain other pathogens,these bacteria are not endemic to the host organism but are ordinarilyintroduced through ingestion of contaminated food or water. Onceintroduced, Salmonellae multiply rapidly in the host and are commonlyassociated with acute intestinal inflammation. Members of this genus areknown to cause such diseases as epidemic meat poisoning, paratyphoidfever, parrot fever, white diarrhea of poultry, fowl typhoid and a swinedisease resembling hog cholera. The gram-negative Salmonellae are alsocharacteristically difficult to destroy, especially in a comparativelydry environment such as animal byproduct meal materials, except by heat.

In the past, various procedures have been suggested for Salmonellacontrol in these meal materials. However, each has proved to have one ormore shortcomings. Sanitation procedures in the processing plant itselfhave failed to achieve 100 percent effectiveness in that even the mostclosely controlled operations have experienced occasional batches ofrecontaminated product. Final heat treatment before shipping cannotprevent recontamination in transit; and both irradiation sterilizationand elevated temperature storage are impractically expensive.

It is therefore a general object of the present invention to provide achemical additive which overcomes the limitations of the prior artprocedures and which is capable of both destroying all viable Salmonellaorganisms that are present and protecting the product against subsequentcontamination.

Another object of the invention is to provide a Salmonella antagonistwhich does not adversely effect the nutritional quality of the treatedfood or feed.

Still another object of the invention is to provide a Salmonellaantagonist which has no adverse effect on the palatability of thetreated food or feed.

A further object of the invention is to provide a Salmonella antagonistwhich is non-toxic to the consuming animal.

A still further object of the invention is to provide a Salmonellaantagonist which is inexpensive and easily applied to the food or feed.

These and other objects and features of the invention will become moreapparent from a consideration of the following descriptions.

DETAILED DESCRIPTION OF THE INVENTION The alkali metal salts ofpropionic acid have been used heretofore as mold inhibitors in thebread-baking industry, but it has been generally recognized thatpropionates have essentially no activity against yeasts and littleaction against bacteria, except for the organism Bacillusmesentericuelwhich causes rope" in bread). The acid itself is usedprincipally as a commerciul csterifying agent in the manufacture ofthermoplastics, solvents, synthetic fruit flavors and perfume bases.

The present invention is based on the discovery that propionic acid hasunexpected antimicrobial activity against Salmonella organisms in animalby-product meal materials and that this activity is markedly enhanced bythe presence of certain other low molecular weight organic acids and/orphosphoric acid. The term animal by-product meal materials is usedherein to describe such edible, nutritious, relatively dry, particulateproducts as meat-and-bone meal, meat meal, tankage, dried blood, poultryby-product meal, fish meal and the like.

The specific nature of the present invention will now be described withreference to certain bacteriological tests and working examples of theinvention.

In the various bacteriological determinations set forth hereinbelow,inoculations were made using Salmonella cultures prepared from fiveSalmonella-Shigella serotypes. The individual cultures for this workwere obtained from the American Type Culture Collection, Rockville, Md.and were coded as follows:

Salmonella senftenberg No. 8400 Salmonella typhi No. 6539 Salmonellacholeraesuis No. 10708 Salmonella typhimurium No. 6994 ShigellaflexneriNo. 9199 In preparing the test cultures, one ml. of a fresh, activeculture of each of the above five serotypes was introduced into ml. ofselenite-cystine broth, and the inoculated broth was then incubated for48 hours at 37 C.

One common measure of the effectiveness of an antimicrobial agentinvolves measuring a zone of inhibition; and such a measure has beenused in certain of the tables set forth hereinafter. The specificprocedure employed in these studies was a modification of the method ofVincent and Vincent (Proc. Soc. Exptl. Biol. Med., 55, 162, 1944), andis described as follows: The mixed Salmonella-Shigella culture wasspread evenly by cotton swabs on the surface of solid, sterile media (20ml.) in petri plates (100 X 15 mm.). The media used was brilliant greenagar. Paper discs 12.7 mm. in diameter were soaked for a few seconds inthe test solutions. Three such discs were suspended by forceps andpressed firmly to the surface of the medium in each petri plate. Theplates were incubated for 24 hours at 37 C., and the inhibitory effectdetermined by measuring the diameter of each zone in which growth didnot occur.

The foregoing sensitivity test was used to determine the comparativebactericidal effect of propionic acid, and the results are set forth inTable I below.

TABLE I Bactericidal Effect Sensitivity Method (Brilliant Green Agar)Individual Acids lactic acid citric acid total plate inhibition The dataset forth in Table 1 points out the superior antimicrobial activity ofpropionic acid. While it is not desired to be limited to any particulartheory, it is believed that undissociated acid is the bactericidallyactive substance and it is consequently the characteristically lowdissociation constant of propionic acid that makes it uniquelyeffective.

A While propionic acid alone possesses bactericidal effectivenessagainst Salmonella organisms, applicants have found that, at least atlow concentrations, it is not always effective in completely destroyingSalmonella bacteria in heavily contaminated meat-and-bone meal. Forexample, the previously described, active, mixed culture ofSalmonelIa-Schigella was added -to sterile meat-and-bone meal at therate of 10 ml. of culture to 100 g. of the meal material. The sterilemeat-and-bone meal was prepared by treating it in an autoclave for 15minutes at 15 p.s.i.g. (120 C.). Enough physiological saline solutionwas added to the inoculated meal material to form a thick slurry and themixture was stirred thoroughly. The mixture was then incubated for 24hours at 37 C. Additional sterile meat-and-bone meal was then addedslowly to the slurry with agitation until the mixture was just moist. Itwas then dried at 37 C. The selected antagonist material was added tothe inoculated meal as a water solution, the solution being slowlypoured into the meal material. The resultant mixture was then agitatedfor a suitable period of time to distribute the antagonist. The resultsare set forth in Table 11 below. It can be observed from the tabulationsthat, while propionic acid showed strong antimicrobial action, completeinhibition was not achieved under the test conditions.

TABLE II The Influence of Different Concentrations of lndividual Acidson Viable Salmonella Organisms in Artificially ContaminatedMeat-and-Bone Meal (Solutions applied at level by weight) In making thequantitative estimations of Salmonella in meat-and-bone meal, three gramsamples, three 1.0 gram samples and three 0.1 gram samples were used forthe following enrichment and subculturing procedure: Each sample wasadded to 225 ml. of 1actose broth which was then incubated for 24 hoursat 37 C. A first 10 m1. portion of this broth was added to ml. oftetrathionate broth which was then incubated for 24 hours at 37 C. Oneloopful of this liquid was then streaked on each of the following:brilliant green agar, Salmonella-Shigella agar, and eosin methylene blueagar. These plates were then incubated for 24 hours at 37 C. and checkedfor Salmonella-like colonies.

A second 10 m1. portion of incubated lactose broth was at the same timeadded to selenite-cystine broth which was then processed like theinoculated tetrathionate broth.

The confirmation tests consisted of the following: Salmonella-suspectcolonies were transferred to link samples of brain-heart infusion brothand the tubes incubated for 4-6 hours at 37 C. A sterile wire needle wasused to inoculate from the brain-heart infusion broth to a tube slant oftriple sugar iron agar. The later tubes were incubated for 24 hours at37 C. and examined for Salmonella-suspect reactions. To the remainingbrain-heart infusion broth there was added an equal volume of 0.6percent formalized physiological saline solution and a check was madefor Salmonella.

H antigens using the Spicer-Edwards rapid l-l identification technique.A check was also made for Salmonella O antigens by performing theagglutination tests using a saline suspension prepared from the triplesugar iron agar slant. Where the isolate agglutinates in the Spicer-Edwards antisera test and in one of the group 0 antisera tests lead tothe conclusion that Salmonella were present, counts were made. Where theresults were doubtful, further biochemical tests (lysine, urea,dulcitol) were conducted using the triple sugar iron slant as theinoculum. The approximate levels of Salmonella organisms in themeat-and-bone meal were estimated using the most Probable Number Tableof l-loskins, (Pub. Health Rep., 49, 396-7, 1934).

As stated hereinabove, the antimicrobial activity of propionic acid ismarkedly enhanced by the presence of certain other low molecular weightorganic acids and/or phosphoric acid. In accordance with the features ofthe present invention, the complementary acid material is selected fromacetic, butyric, citric, lactic and phosphoric acids; and in a preferredform of the invention two parts of propionic acid are combined with twoparts of acetic or lactic acid and with one part of phosphoric, butyricor citric acid.

The effectiveness of the acid compositions of the invention isillustrated in Tables Ill and IV, the specific combination of acidsemployed in the studies which resulted in the data shown in Table 111being set forth in Table V.

TABLE lll Influence of Different Concentration of Acid Mixtures onViable Salmonella Organisms in Artificially Contaminated Meat-and-BoneMeal 1,700 1,900 1,600 2,000 1,800 Formula 1 30 30 20 20 2 TABLE IVBactericidal Effect Sensitivity Method (Brilliant Green Agar) AcidCombinations Composition (per cent by weight) propionic acetic butyricphosphoric citric lactic water rmulaA l0 5 10 75 F0- rmulaB l0 l0 5 75F0- rmulaC l0 l0 5 75 The data set forth in Table II] was accumulatedusing the procedures previously described with reference to Table llwhereas the data shown in Table IV was generated using the procedurespreviously described with reference to Table l.

Table lll clearly indicates the bactericidal effect of compositionscomprising propionic acid and combinations of acetic, butyric andphosphoric acids, this effectiveness being rapid and substantiallycomplete in additions to meat-and-bone meal at the 5 percent level byweight. Compositions incorporating propionic acid and combinations oflactic and citric acids are also highly effective as is shown by thedata in Table IV.

TABLE VI Influence of Formula A on Salmonella Counts in ArtificiallyContaminated Meat Meal Concentration of Salmonella Count Per GramFormula A initial 24 hrs. 48 hrs. 72 hrs. 1 week 0 (Control) 1,900 1,7001,800 2,000 2,100 l 40 20 30 20 20 3 30 30 20 l0 l0 5 0 0 0 0 0 10 0 0 00 0 A consideration of Tables Ill and VI shows that the antimicrobialcompositions of the invention have effectiveness at even the ldpercentlevel when a 25 percent active material is use for the treatment. Thesetables further indicate that the acid compositions of the inventionachieve complete destruction of Salmonella organisms at between the 3and 5 percent levels of addition, that is, when approximately 1% percentof active material is introduced into the meal.

Other studies have established that the bactericidal effect of thematerials of the instant invention is not the result of a merealteration in the acidity level. For example, the pH of treatedmeat-and-bone meal is approximately 4.5; and acidity alone in the rangeof pH 2.5 to 8.5 has been found to have no effect on the growth ofSalmonellae.

Feeding trials with chicks have shown that acid treatment ofmeat-and-bone meal in accordance with the present invention has noadverse effect either on the acceptability of rations containing themeal or on the nutritional efficiency of such rations. Furthermore, theacid treatment of the invention produces no unacceptable effect on themoisture, fat or protein content of the meal with additions as high as 1percent of active material. Residual effectiveness has been establishedby studies in which attempts to contaminate previously treated mealfailed.

Incorporation of the bactericides of the invention may be achieved bythe spraying of water solutions of the ingredients into an activelymixing meal, in addition to the mixing procedures described previously.

While particular embodiments of the invention have been describedhereinabove, it should be understood, of course, that the invention isnot limited thereto since many modifications may be made; and it istherefore contemplated to cover by the present application, any suchmodifications as fall within the true spirit and scope of the appendedclaims.

The invention is claimed as follows:

1. A salmonellacide composition comprising two parts of propionic acid,two parts of acetic acid and one part of phosphoric acid.

2. A salmonellacide composition comprising two parts of acetic acid andone part of butyric acid.

2. A salmonellacide composition comprising two parts of acetic acid andone part of butyric acid.